1. Field of the Invention
This invention relates to the identification and isolation in substantially pure form of an antigenic protein material from mature styles of self-incompatible plants, particularly members of the Solanaceae and the Cruciferae families, as exemplified by Nicotiana alata, an ornamental tobacco, Lycopersicon peruvianum, a wild tomato, and Brassica oleracea, Brussel sprout. Studies of this style-specific material indicate that it corresponds to the self-incompatibility genotype of such self-incompatible plants, and is the product of the S-gene which controls self-incompatibility.
Accordingly, this material has potential for use in control of pollentube growth, for example, as a natural gametocide to control, induce, or promote self-incompatibility and interspecific incompatibility. The S-gene and its product can also be used in genetic manipulation of plants to create self-incompatible cultivars. Plants engineered in this way will be valuable for the economic production of hybrid seed. In summary, the utility of this material will be in (i) production of selfed seed in out-breeding crops; (ii) creation of new interspecific hybrid crops; and (iii) generation of methods for economic production of hybrid seed in currently self-compatible crops.
2. Description of the Related Art
Many plant species, including Nicotiana alata and Lycopersicon peruvianum, are self-incompatible, that is, they cannot be fertilized by pollen from themselves or by that of a plant of the same S- (or self-incompatibility) genotype. The molecular basis of self-incompatibility is believed to arise from the presence of S-gene protein material in the mature styles of the plants; in particular, as exemplified by N. alata and L. peruvianum, this S-gene protein material has now been shown to be present in extracts of plant styles at the developmental stages of buds at first show of petal color, and at the subsequent stages of maturation of open but immature flowers, and flowers having mature glistening styles. On the other hand, the S-gene protein material is not present in the earlier developmental stages of green bud and elongated bud.
In plants exhibiting self-incompatibility, where the same S-gene is present in both the pollen and the pistil, pollen tube growth is arrested in the style of the plant and fertilization fails to occur. In both incompatible and compatible pollinations, the initial events are apparently identical. The pollen tubes germinate and grow extracellularly between the stigma papillae and then between the cells of the transmitting tissue of the style, which are joined by plasmodesmata into vertical files. At maturity, the extracellular material becomes progressively less viscous so that the pollen tubes grow through an amorphous fluid matrix between files of cells. At some point within the style, the growth of incompatible tubes is arrested. The precise zone of the style at which this happens varies with the ambient temperature. The behavior of the pollen tubes during growth indicates that the reaction may involve contact between the growing pollen tube and the extracellular components of the style, in particular contact of the growing tube with S-gene protein material as discussed above.
For general reviews of self-incompatibility, see de Nettancourt (1977), Incompatibility in Angiosperms. Springer-Verlag, Berlin; Heslop-Harrison (1978), Proc. Roy. Soc. London B, 202:73; Lewis (1979) N. Z. J. Bot. 17:637; Pandey (1979), N.Z. J. Bot. 17:645 and Mulcahy (1983) Science 220:1247. Selfincompatibility is defined as the inability of female hermaphrodite seed plants to produce zygotes after self-pollination. Two types of self-incompatibility, gametophytic and sporophytic, are recognized. Gametophytic incompatibility is most common and in many cases is controlled by a single nuclear gene locus (S-locus) with multiple alleles. Pollen expresses its haploid S-genotype and matings are incompatible if the S-allele expressed is the same as either of the S-alleles expressed in the diploid tissue of the pistil. During both incompatible and compatible matings, pollen tubes germinate and grow through the stigma into the transmitting tissue of the style. Tube growth from incompatible pollen grains is arrested in the upper third of the style.
In sporophytic incompatibility, pollen behavior is determined by the genotype of the pollen-producing plant. If either of the two S-alleles in the pollen parent is also present in the style, pollen tube growth in inhibited. Unlike gametophytic systems, inhibition usually occurs at the stigma surface and not in the style. In sporophytic incompatibility, S-protein may be concentrated at or near the stigma surface. The gametophytic polyallelic system is considered to be the ancestral form of self-incompatibility in flowering plants, with the sporophytic system being derived from it (de Nettancourt 1977). The products of the S-gene in the two systems are considered to be structurally related.
There are five species of gametophytically self-incompatible plants and two species of sporophytically incompatible plants in which differences in style or stigma proteins apparently related to S-genotype have been detected electrophoretically or immunologically. In N. alata, an association between specific bands and three S-allele groups was demonstrated by isoelectric focussing of stylar extracts (Bredemeijer and Blaas (1981) Theor. Appl. Genet. 59:185). Two major antigenic components have been identified in mature styles of Prunus avium, one of which (S-antigen) was specific to a particular S-allele group (Raff, et al. (1981) Planta 153:125; and Mau, et al. (1982) Planta 156:505). The antigenic component, a glycoprotein, was isolated by gel-filtration and ion-exchange chromatography. Two dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) resolved two components (molecular weights 37,000 and 39,000, respectively) which could correspond to the S.sub.3 and S.sub.4 products in the diploid stylar material of cultivars of S.sub.3 S.sub.4 genotype. The material was a potent inhibitor of the in vitro growth of pollen tubes from a S.sub.3 S.sub.4 cultivar (Williams, et al. (1982) Planta 156:517). Stylar components which correspond to the S-allele group and are effective antigens in rabbits were detected in Petunia hybrida as reported by Linskens (1960), Z. Bot. 48:126. Four of the stylar proteins of Lilium longiflorum separated by electrofocussing reportedly showed distinct differences before and after heat treatment of the styles at 50.degree. C. The S-gene product is thought to be heat labile because self-incompatibility can be broken by heat-treatment at 50.degree. C; hence the bands lost during heat treatment may have corresponded to S-gene products (Dickinson, et al. (1982), Proc. Roy. Soc. London B. 215:45). A protein of molecular weight 24,000 isolated from stylar canal fluid of Trifolium pratense by Heslop-Harrison (1982) Ann. Bot. 49:729, may be involved in self-incompatibility.
A glycoprotein corresponding to genotype S.sub.7 of Brassica campestris has been isolated from extracts of stigmas by gel-filtration followed by affinity chromatography on Con-A-Sepharose (Trademark, Pharmacia, Inc. Uppsala, Sweden) then isoelectric focussing, Nishio and Hinata (1979) Jap. J. Genet. 54:307. The protein had a pI of 5.7, molecular weight (by SDS-PAGE) of 57,000 and contained protein and carbohydrate in the ratio 1:1.2. The protein was not present in styles or ovaries. Nishio and Hinata (1982) Genetics 100:641, used similar techniques to isolate S-specific glycoproteins from stigma extracts of Brassica oleracea plants homozygous for S-alleles S.sub.39, S.sub.22 and S.sub.7. The glycoproteins had high pI's (10.3, 11.1 and 10.6 respectively) and contained protein and carbohydrate in the ratios of 1:0.05 (S.sub.7 and S.sub.22) and 1:0.2 (S.sub.39) The apparent molecular weight (by SDS-PAGE) of glycoproteins S.sub.7 and S.sub.39 was 57,000. Glycoprotein S.sub.22 was apparently heterogeneous; two dimensional SDS-PAGE resolved two components of molecular weights 60,000 and 65,000. Antisera raised to each isolated S-specific glycoprotein not only precipitated its homologous glycoprotein but also the other two S-specific glycoproteins of B. oleracea and the S.sub.7 -specific glycoprotein of B. campestris (Hinata et al (1982) Genetics 100:649). A similar S-specific glycoprotein was isolated by Ferrari, et al. (1981) Plant Physiol. 67:270, from a stigma extract of B. oleracea using different techniques. Pretreatment of S.sub.2 S.sub.2 pollen with the glycoprotein prevented the pollen germinating on normally compatible stigmas, indicating that the preparation was biologically active. Another partial purification was reported by Ferrari et al. (1981) using sucrose gradient sedimentation and double diffusion tests in gels in which the proteins were identified by Coomassie Blue staining.
Peroxidase isoenzymes extracted from styles of N. alata were reported to be S-gene specific by Pandey (1967), Nature 213:669. Subsequently, Bredemeijer and Blass (1980) Theor. Appl. Genet. 57:119, using the same species were unable to find any relationship between S-genotype and the peroxidase isozyme pattern and concluded that the peroxidases were coded by genes closely linked to the S-locus. It had been suggested that one of the isoenzymes in N. alata styles is involved in pollen tube growth (Bredemeijer and Blass (1975) Acta Bot. Naerl. 24:37), but it was subsequently found that this isoenzyme occurs in the cortex of the style, but is absent from the transmitting tissue, the site of pollen tube growth (Bredemeijer (1979) Acta Bot. Neerl. 28:197). Nasrallah, et al. (1970) Heredity 25:23, and Nishio and Hinata (1977) Heredity 38:391, were unable to detect any relationship between S-genotype and the pattern of peroxidase isoenzymes in stigma extract of B. oleracea. Therefore, the possible role, if any, of peroxidase isoenzymes in the incompatibility reaction is unclear.
S-L. Mau et al. (1986) in an article entitled "Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility", Planta 169:184-191, published less than a year prior to the filing date hereof, discuss some of the work on which this application is based.